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Microbial Interactions in AquacultureDisease Control in Shrimp Aquaculture with Probiotic Bacteria
Biomanagement Systems Pty. Ltd., 315 Main Road, Wellington Point. Queensland 4160 Australia,and Department of Chemical Engineering, The University of Queensland. Qld. 4072 Australia.ABSTRACT
Shrimp aquaculture production in much of the world is depressed by disease, particularlycaused by luminous Vibrio and/or viruses. Antibiotics, which have been used in largequantities, are in many cases ineffective, or result in increases in virulence of pathogensand, furthermore, are cause for concern in promoting transfer of antibiotic resistance tohuman pathogens. Probiotic technology provides a solution to these problems. Themicrobial species composition in hatchery tanks or large aquaculture ponds can be changedby adding selected bacterial species to displace deleterious normal bacteria. Virulence ofluminous Vibrio species can be controlled in this manner. Abundance of luminous Vibriostrains decreased in ponds and tanks where specially selected, probiotic strains of Bacillusspecies were added. A farm on Negros, in the Philippines, which had been devastated byluminous Vibrio disease while using heavy doses of antibiotics in feed, achieved survivalof 80-100% of shrimp in all ponds treated with probiotics.Introduction
The UN FAO estimates that half of the world’s seafood demand will be met by aquaculturein 2020, as wild capture fisheries are overexploited and are in decline. Shrimp (or prawn)culture is widespread throughout the tropical world. It is in an industry set for a period ofstrongly growing demand, and is currently worth around US$10 billion. Penaeusmonodon, the black tiger shrimp, is the most widely cultured species.
In much of the world, however, the shrimp aquaculture industry is beset by disease,
mostly due to bacteria (especially the luminous Vibrio harveyi) and viruses. The highdensity of animals in hatchery tanks and ponds is conducive to the spread of pathogens,and the aquatic environment, with regular applications of protein-rich feed, is ideal forculturing bacteria.
I feel these problems have been exacerbated because the interactions of microbes,
animals and their environment at intensive production scales have been consideredprimarily from a clinical pathology perspective. That is, the disease was treated rather thanthe underlying cause. When pathogenic bacteria or viruses are detected, farmers applyantimicrobial compounds to the feed and water. Many farmers also use antibiotics asprophylactics in large quantities, even when pathogens are not evident. This has lead to anincrease in vibrios, and presumably other bacteria, having multiple antibiotic resistanceand to an increase in more virulent pathogens. Many of the pathogens appear to havemutated to more virulent forms than were present a decade ago, and thus even when theshrimps are not stressed by poor water quality they succumb to attack. Thus I feel that theincidence of disease has been exacerbated by the actions of the shrimp farmers.Microbial Biosystems: New FrontiersProceedings of the 8th International Symposium on Microbial EcologyBell CR, Brylinsky M, Johnson-Green P (eds)Atlantic Canada Society for Microbial Ecology, Halifax, Canada, 1999.Microbial Interactions in Aquaculture
The solution lies in the field of microbial ecology, not in the field of pharmacology, i.e.
in developing new antibiotics or vaccines [5]. Shrimp farmers have to learn to live with acomplex community of microbes and manage them. The use of beneficial bacteria(probiotics) to displace pathogenic bacteria by competitive processes is a better remedythan administering antibiotics. And it works!
The microbial species composition in aquaculture ponds can be changed by adding
selected species to displace deleterious common bacteria. Success depends upon definingthe ecological process or processes to be changed, the types of deleterious species that aredominant and the desirable alternative species or strains of bacteria that could be added.Competitive exclusion is one of the ecological processes that allows manipulation of thebacterial species composition in the water, sediment and animal guts.Antimicrobials and PathogensVibrio spp., especially the luminous V.harveyi, have been implicated as the main bacterialpathogens of shrimps [2]. Antibiotics have been used in attempts to control these bacteria,but their efficacy is now, in general, very poor. In the Philippines, luminous Vibrio diseasecaused a major loss in shrimp production in 1996, and many farms have ceased to produceshrimps because survival was so poor. The Vibrio species were resistant to everyantibiotic used, including chloramphenicol, furazolidone, oxytetracycline, andstreptomycin, and were more virulent than in previous years.
In Thailand this year, a farmer who was using colloidal silver in all feeds experienced a
large increase in mortality from vibriosis. This was managed for a while with large dosesof norfloxacin in all feeds. However, when it was stopped all shrimps died within 2 days.Clearly, a highly virulent strain of luminous Vibrio had developed in response to the use ofthe silver and antibiotics.
Chlorine is widely used in hatcheries and ponds, but its use stimulates the development
of multiple antibiotic resistance genes in bacteria [8]. Some farmers in Thailand havereported that when chlorine is used in ponds to kill zooplankton before stocking shrimp,there is a rapid increase in Vibrioharveyi numbers after the chlorine is removed. This is tobe expected as marine vibrios have very fast growth rates, and the chlorine treatment willlower the numbers of competitors for nutrients and kill algae, thus increasing foodresources. It is likely, therefore, that the vibrios surviving after chlorine treatment are notonly more resistant to antibiotics, but are also pathogenic. Thus the problems have beenexacerbated by the use of antimicrobial compounds.
If antibiotics or disinfectants are used to kill bacteria, some bacteria will survive, either
strains of the pathogen or others, because they carry genes for resistance. These will thengrow rapidly because their competitors are removed. Any virulent pathogens that re-enterthe pond or hatchery tank, perhaps from within biofilms in water pipes or in the guts ofanimals, can then exchange genes with the resistant bacteria and survive further doses ofantibiotic. Thus, antibiotic-resistant strains of pathogens evolve rapidly.
The transfer of resistance to human pathogens and gut bacteria is of major concern.
Such transfers probably happen easily and often, as discussed by Salyers [10]. Resistanceplasmids encoding for many antibiotic resistance genes were transferred betweenpathogenic and non-pathogenic Gram negative bacteria in several environments includingsea water. In the presence of tetracycline concentrations that were not high enough to killthe bacteria, the rate of gene transfer between Vibrio cholerae and Aeromonas salmonicidaincreased 100 times [4].Microbial Interactions in Aquaculture
This work raises questions not only about the use of antibiotics in aquaculture, but about
the use of bacteria closely related to pathogenic species as probiotics. Not onlyantimicrobial resistance genes, but also genes for virulence can be transferred by Rplasmids and transposons. As the R plasmids can transfer genes between widely differentbacteria in the Gram negative group, it would be potentially dangerous to use Vibrio orPseudomonas, for example, as probiotics.
Throughout Asia, shrimp farmers use antibiotics in large quantities. Warehouses
supplying the industry in all the major centres sell a range of antibiotics in containers of500 g or more in size. The antibiotics in current use include fluoroquinolones especiallynorfloxacin and enrofloxacin, furazolidone, oxolinic acid, oxytetracycline, trimethoprimand sulphadiazine. It is difficult to find out just how much antibiotic use there is in theindustry, but it is possible to make an estimate from feed usage and production. In 1994Thailand produced about 250,000 tonnes (a quarter of the world production) of farmedshrimps, which consumed about 500,000 - 600,000 tonnes of feed. With the diseaseproblems, shrimp production last year was down to 150,000 tonnes. For each crop atsemi-intensive to intensive scales of production, farmers use 5 - 10 g antibiotics per kgfeed at least once per day at weekly intervals; some use them for more extensive periods.Thus antibiotics would be used in about 10% of feed. It is possible, therefore, that theantibiotic usage in shrimp farm production in Thailand in 1994 was as much as 500 - 600tonnes, assuming all farmers used them — and this does not include that used in hatcheriesfor fry production.
As much of this will end up producing bacteria with multiple antibiotic resistance in
farm effluents that then contaminate coastal waters, the potential impact on human healthis significant. This problem was discussed by Austin in 1983 [1] with reference to fishfarming, but it has become far worse with the major increase in shrimp farming that hasoccurred since then.Probiotic Bacteria
The use of beneficial bacteria (probiotics) to displace pathogens by competitive processesis being used in the animal industry as a better remedy than administering antibiotics and isnow gaining acceptance for the control of pathogens in aquaculture [3]. The term“probiotic” has been defined as: “a probiotic is a mono- or mixed culture of livemicroorganisms that, applied to animal or man, affect beneficially the host by improvingthe properties of the indigenous microflora” [3]. In this discussion, the authors consideredonly human and land farm animals. In extending their definition to aquaculture, I suggestthat it also applies to the addition of live, naturally-occurring bacteria to tanks and ponds inwhich the animals live, because these bacteria modify the bacterial composition of thewater and sediment. The health of animals is thus improved by the removal, or decrease inpopulation density, of pathogens and by improving water quality through the more rapiddegradation of waste organic matter.
Unlike land animals, aquatic farmed animals are surrounded by a milieu that supports
opportunistic pathogens independently of the host animal, and so the pathogens can reachhigh abundance around the animal. Vibrio grow attached to algae, and may reach highpopulation densities after being ingested with the algae and then excreted with lysed algaein faecal pellets by zooplankton; they are gut bacteria in fish and shrimps as well aszooplankton [7]. In aquaculture ponds, where animal and algal population densities arevery high, Vibrio numbers are also high compared to the open sea. The onset of shrimp
Microbial Interactions in Aquaculture
disease due to exposure to high numbers of Vibrio, especially when pathogenicity hasincreased by overuse of antimicrobial compounds indicates that a defense is needed.
The species composition of a microbial community, such as that in a pond, will be
determined partly by stochastic phenomena, that is, chance, and partly by deterministic andpredictable factors that allow one species to grow and divide more rapidly than others, andthus dominate numerically. Chance favours those organisms that happen to be in the rightplace at the right time to respond to a sudden increase in nutrients, e.g. from the lysis ofalgal cells or the decomposition of feed pellets that fall around them. The farmer canmanipulate the species composition by seeding large numbers of desirable strains ofbacteria or algae; in other words, by giving chance a helping hand.
Competitive exclusion is one of the ecological processes that can be manipulated to
modify the species composition of a soil or water body or other microbial environment.Small changes in factors that affect growth or mortality rates will lead to changes inspecies dominance. We are still a long way from knowing all the factors that controlgrowth rates of particular species. The complete species composition in naturalenvironments is largely unknown, but enough is known to argue that it is possible tochange species composition by making use of competitive exclusion principles [11]. Thusbacteria can compete by secreting antimicrobial compounds that do not necessarily kill alltheir competitors, but increase mortality rates just enough to tip the balance in resourceutilization. For example, if a Bacillus strain were to produce an antibiotic that inhibited aVibrio, then the Vibrio’s mortality rate would increase, shifting the dominance to theBacillus, even if the antibiotic were not produced at high enough concentration to kill all ormost Vibrio cells directly.
Microbial ecology and biotechnologies have advanced in the last decade, to the point
that commercial products and technologies are available for treating large areas of waterand land to enhance population densities of particular microbial species or biochemicalactivities. The practice of bioremediation (or bioaugmentation) is applied in many areas,but success varies greatly, depending on the nature of the products used and the technicalinformation available to the end user. The bacteria that are added must be selected forspecific functions that are amenable to bioremediation, and be added at a high enoughpopulation density, and under the right environmental conditions, to achieve the desiredoutcomes. Bioaugmentation and the use of probiotics are significant management tools foraquaculture, but their efficacy depends on understanding the nature of competition betweenparticular species or strains of bacteria. They rely on the same concepts that are usedsuccessfully for soil bioremediation and probiotic usage in the animal industry.
Probiotics such as the Gram positive Bacillus offer an alternative to antibiotic therapy
for sustainable aquaculture. Bacillus species are commonly found in marine sediments andtherefore are naturally ingested by animals such as shrimps that feed in or on the sediment.An advantage of using Bacillus species is that they are unlikely to use genes for antibioticresistance or virulence from the vibrios or related Gram negative bacteria. There arebarriers at the transcriptional and translational levels to the expression of genes fromplasmid, phage and chromosomal DNA of E. coli in B. subtilis [9].Probiotic Applications in Aquaculture
Bacterial species composition in shrimp ponds, which are large water bodies up to ahectare or more in size, hatchery tanks and shrimp guts can easily be changed and thus
Microbial Interactions in Aquaculture
result in an improvement in shrimp production. In particular, luminous Vibrio can becontrolled in this manner. To my knowledge, there has not been any rigorous study madeof Vibrio populations in shrimp on farms, in relation to antibiotic or probiotic usage. Thusthe data referred to here are given as examples of what has been observed, but theconclusions need to be substantiated.
An example is the Viveros farm in Negros, where losses from luminous Vibrio had been
catastrophic, even though antibiotics were used in the feed and gave protection some of thetime. Luminous Vibrio abundance in the pond waters of that region were often as high as103 to 104 per ml within 2 - 3 weeks of filling and fertilizing ponds. However, when theprobiotic bacteria were used, no disease was experienced and indeed survival was veryhigh (80-100%), even in the presence of luminous Vibrio species (Tables 1, 2) [6].Table 1. Shrimp production on a farm in Negros, The Philippines, with BiomanagementSystems’ probiotic bacterial technology, compared to controls with antibiotics [data frommany other control ponds that collapsed after 30-60 days are not shown]. Ponds wereharvested in December 1996/January 1997. All shrimps died overnight in pond 6 fromvibriosis, which was a considerable loss at 120 days of culture.Table 2. Second crop from a farm in Negros (stocked April 1997, harvested September) in5 of the ponds referred to in Table 1 above.
In several ponds in the Philippines, luminous Vibrio numbers in the hepatopancreas of
shrimps fell from around 1 x 104 per gut when antibiotics were used in the feed, to zerowhen probiotics were applied to the pond (Figure 1). The shrimp gut flora was oftendominated by Vibrio species that were sucrose negative (green on TCBS agar) andluminous in the presence of antibiotic treatment, whereas sucrose positive strains (yellowcolonies) were usually more abundant or equally abundant when probiotics were used (Fig.1). Many farmers now realize that they cannot solve the vibriosis disease problem byusing antibiotics.Microbial Interactions in AquacultureFig. 1. Total and luminous Vibrio in midgut (hepatopancreas) of shrimps in Pond 25 , Roxas, Philippines.Luminous Vibrio were often abundant in the presence of antibiotics: i.e. they were resistant and shrimpmortality was high, although sometimes they were sensitive, e.g. at 132 Days of Culture, when the antibioticswere changed. After Day 160, when the probiotic Pondpro-VC was added daily to the pond, luminous Vibriowere eliminated from the gut. (Data were kindly supplied by AA Exico, Roxas City.)
Luminous Vibrio were completely eliminated from the water column and from the
sediment of ponds in Indonesia when probiotic strains selected for their direct inhibitoryeffect were used [11]. In contrast, Vibrio numbers increased markedly from around 20 toover 200 cfu/ml in shrimp ponds where antibiotics were used in the feed. Survival, andthus production, was high in all ponds where probiotics were used.
These data show that the disease problems can be overcome by applying probiotic
biotechnology, which is an application of microbial ecology. It makes use of the naturalmechanisms by which bacteria compete against each other. In other words, shrimp farmerswho learn to farm microorganisms will be far more likely to achieve successful harvests.
With the right combination of bacteria and aeration, water exchange can be minimized
and water can be recycled between crops, thus lessening environmental impacts and thelikelihood of introducing pathogens. The transfer of antibiotic resistance to humanpathogenic bacteria, which is exacerbated by the abuse of antibiotics in the aquacultureindustry, will decrease.References
1. Austin, B (1993) Environmental issues in the control of bacterial diseases of farmed
fish. In: Environment and Aquaculture in Developing Countries. Pullin, RSV.Rosenthal, H, Maclean, JL (eds). ICLARM Conference Proceedings 31. ICLARM,Manila. pp 237-251
2. Baticados, MCL, Lavilla-Pitogo, CR, Cruz-Lacierda, ER, de la Pena, LD, Sunaz, NA
(1990) Studies on the chemical control of luminous bacteria Vibrio harveyi and V.splendidus isolated from diseased Penaeus monodon larvae and rearing water. DisAquat Org 9: 133-139
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3. Havenaar, R, Ten Brink, B, Huis in’t Veld, J H J (1992) Selection of strains for
probiotic use. In: R. Fuller (ed), Probiotics: the scientific basis, Chapman and Hall,London. pp 209-224.
4. Kruse, H, Sørum, H (1994) Transfer of multiple drug resistance plasmids between
bacteria of diverse origins in natural environments. Appl Environ Microbiol 60: 4015-4021
5. Moriarty, DJW (1997) The role of microorganisms in aquaculture ponds. Aquaculture
6. Moriarty, DJW (1998) Control of luminous Vibrio species in penaeid aquaculture
7. Moriarty, D.J.W. 1990. Interactions of microorganisms and aquatic animals,
particularly the nutritional role of the gut flora. In: R. Lésel (ed.), Microbiology inPoecilotherms: Proceedings of the International Symposium on Microbiology inPoecilotherms. Elsevier Science Publishers, B.V. pp. 217- 222.
8. Murray, G. E., Tobin, R. S., Junkins, B., Kushner, D. J. (1984) Effect of chlorination
on antibiotic resistance profiles of sewage related bacteria. Appl Environ Microbiol.48: 73-77
9. Rabinowitz JC, Roberts, M (1986) Translational barriers limiting expression of E. coli
genes in Bacillus and other Gram-positive organisms. In: Levy, S.B, Novick, R.P (eds)Banbury Report 24: Antibiotic Resistance Genes: Ecology, Transfer and Expression.Cold Spring Harbour Laboratory pp297-312
10. Salyers, A A (1995) Antibiotic resistance transfer in the mammalian intestinal tract:
implications for human health, food safety and biotechnology. Springer-Verlag, NewYork. pp 109-136.
11. Smith, V H (1993) Implications of resource-ratio theory for microbial ecology. Limnol

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